Salt flat microbial diversity and dynamics across salinity gradient

Sabkhas are hypersaline, mineral-rich, supratidal mudflats that harbor microbes that are adapted to high salt concentration. Sabkha microbial diversity is generally studied for their community composition, but less is known about their genetic structure and heterogeneity. In this study, we analyzed a coastal sabkha for its microbial composition using 16S rDNA and whole metagenome, as well as for its population genetic structure. Our 16S rDNA analysis show high alpha diversity in both inner and edge sabkha than outer sabkha. Beta diversity result showed similar kind of microbial composition between inner and edge sabkha, while outer sabkha samples show different microbial composition. At phylum level, Bacteroidetes (~ 22 to 34%), Euryarchaeota (~ 18 to ~ 30%), unclassified bacteria (~ 24 to ~ 35%), Actinobacteria (~ 0.01 to ~ 11%) and Cyanobacteria (less than 1%) are predominantly found in both inside and edge sabkha regions, whereas Proteobacteria (~ 92 to ~ 97%) and Parcubacteria (~ 1 to ~ 2%) are predominately found in outer sabkha. Our 225 metagenomes assembly from this study showed similar bacterial community profile as observed in 16S rDNA-based analysis. From the assembled genomes, we found important genes that are involved in biogeochemical cycles and secondary metabolite biosynthesis. We observed a dynamic, thriving ecosystem that engages in metabolic activity that shapes biogeochemical structure via carbon fixation, nitrogen, and sulfur cycling. Our results show varying degrees of horizontal gene transfers (HGT) and homologous recombination, which correlates with the observed high diversity for these populations. Moreover, our pairwise population differentiation (Fst) for the abundance of species across the salinity gradient of sabkhas identified genes with strong allelic differentiation, lower diversity and elevated nonsynonymous to synonymous ratio of variants, which suggest selective sweeps for those gene variants. We conclude that the process of HGT, combined with recombination and gene specific selection, constitute the driver of genetic variation in bacterial population along a salinity gradient in the unique sabkha ecosystem.

. Heatmap of ANI percent identity for the 225 bins described genomes. Cells in the heatmap correspond to 97 % ANI sequence identity are coloured in red. Blue cells correspond to genomes that don't belong to the same species. Color intensity fades as the comparaison approach 97 % ANI sequence identity. Colour bar above and left of the heatmap is indicating the source species assignments for each bin in the analysis. Hierarchical clustering was done suing simple linkage of ANI percent identity. The bottom histogram shows the % ANI identidy and counts of MAG for indentical and different genomes. There is no signi cant correlation (pearson, R=-0.12, p=0.53) between them, suggesting that diversity seen in our sabkha is not correlated with abundance.      Figure   H2S (− 2)  Figure   H2S (− 2) 0.09 S1 S12 S13 S14 S17 S18 S3 S8 Pi Sites Supplementary Figure 8. Plot showing nucleotide diversity that persist at finer scale per site across the sabkha inside and outside study region. Higher diversity was significant for Halobacteriales and Pseudomonadales populations on the inside and Rhizobiales and Pseudomonadales from outside (pairwise t-test Bonferroni p< 0.05).  Nucleotide Diversity SNPs Nucleotide Diversity  Figure 9. Nucleotide diversity was measured using the total number of SNPs/Mbp as metrics methods, which is less sensitive to changes in coverage and is done both for single site and averaged across loci, where also frequencies of SNPs is included not only SNPs by itself.     A) The production of IAA bacterial species isolated from Sabkha B) ACC deaminase produced by actinomycete S. mutabilis (left), while the control plate on (Right). C) ACC deaminase produced by different bacteria isolated from Sabka soil, on the right were ACC producers while on the left (Control). D) The growth of Streptomyces mutabilis on SNA medium amended with different concentrations of NaCl: 1.2%, 2.4%, 3.6%, and 4. 8% while the above is SNA without salt (control).

Isolation of Bacteria and Actinomycetes from coastal sabkha soil
Soil samples limited to a 3 cm-depth were obtained from costal sabkha in United Arab Emirates (UAE) with clean spade in clean plastic bags. These samples represented three different areas, inner, middle, and outer region of the area. Soil samples were air-dried for 5 days at 28ºC to reduce the number of the contaminant bacteria 1 . Passed through a 5-mm mesh sieve to remove small stones and big soil particles. Fine soil stored in sterile screw-capped jars at 25ºC in the dark, for a week prior to microbiological processing. Bacteria and actinomycete were isolated using the soil dilution plate method 2 on inorganic salt starch agar (ISSA) 3 amended with cycloheximide and nystatin (each 50µg per ml; Sigma -Aldrich) with specific soil pretreatments 4 . Briefly, the soil pre-treatment involved preparing serial dilutions of the soil suspension by suspending the sample in 6% yeast extract (YE) (Lab M limited) and 0.05% sodium dodecyl sulfate (SDS) (Sigma -Aldrich) for 20 min at 40ºC, and diluting with water to remove other factors promoting bacterial growth or injurious to germinating actinomycete propagules. The YE and SDS were included to increase and decrease the numbers actinomycete and bacteria, respectively 5 . Five replicate plates were used per dilution, which were incubated at 28ºC in dark for seven days. Actinomycete colony was transferred onto oatmeal agar plates supplemented with 0.1% yeast extract (OMYEA) 3 . While the bacterial isolates were transferred onto nutrient agar plates (NA).
We have obtained from the three different areas mentioned before only one actinomycete while eight different bacterial isolates. All were characterized and identified by using 16S rRNA gene amplification (Supplementary Table 15). All isolated and identified actinomycete and bacteria were subjected to different biological characters (Supplementary Fig 23).

Production of Indole Acetic Acid (IAA)
Nutrient Agar broth used for bacterial isolates and Starch Nitrate for the actinomycete amended with 5-mmole L -tryptophan was added for each and incubated at 37ºC for bacteria and 28ºC for actinomycete with shaking 180 rpm for 48 h. One ml of culture was centrifuged at 4000 rpm for 10 min and the supernatant was separated. To the supernatant, 2 ml of Salkowiski reagent was added followed by incubation at room temperature under dark conditions. Absorbance of the pink color developed was read at 530 nm. The amount of IAA produced by each isolates was expressed in µg/ml cell protein ( Supplementary Fig 23 A) 6 . The production of IAA for bacterial species isolated from Sabkha was measured using optical Density readings at 530 nm for IAA produced by bacterial Isolates and actinomycetes from Sabka (Supplementary Table 15).

Phosphate Solubilization
The isolated bacteria from Sabhka (1 to 9) were spot inoculated onto Pikovaskya's agar medium (Hi -media) amended with 2% (w/v) tricalcium phosphate (TCP) and incubated at 28ºC for 5 days. The development of a clear zone around the colonies was considered as positive phosphate solubilizes (Supplementary Table 15).

In Vitro Assay for Stress Tolerance in Response to Salinity
In order to evaluate salt tolerance characterization, the growth of the endophytic bacteria was observed at 28ºC for 72 h in LB agar medium, supplemented with 2 -10% Nacl concentration 7 . Moreover, the bacterial suspension of all isolates was used to inoculate sterilized conical flask with nutrient broth medium supplemented with 2 -10% Nacl, and incubated in a shaker (180 rpm) for 72 h. while for actinomycete Starch Nitrate Agar (SNA) supplemented with the same concentrations of Nacl was used, for control each isolate only nutrient broth medium was used. Then optical density was measured at 600 nm for all sodium chloride concentration compared to the control. (Supplementary Table 15, Supplementary Fig 23 D)

Characterization and Estimation of ACC Deaminase Activity of bacterial isolated from Sabkha soil
The isolated bacteria were screened for ACC deaminase activity on sterile minimal DF (Dworkin and Foster) salts media (DF salts per liter: 4.0 g KH2PO4, 6.0 g Na2HPO4, 0.2 g MgSO4.7H2O, 2.0 g glucose. 2.0 g citric acid and 2.0 gluconic acid with trace elements: 1 mg FeSO4.7H2O, 10 mg H3PO3, 11.19 mg MnSO4.H2O, 124.6 mg ZnSO4.7H2O, 78.22 mg CuSO4.5H2O, 10 mg MoO3, pH 7.2) amended with 3 mM ACC (Sigma -Aldrich), while for control the media was supplemented with (NH4) 2SO4 as a sole nitrogen source instead of ACC 8,9 . The inoculated plates were incubated at 28ºC for 3 days and growth was monitored on a daily basis. Colonies growing on the plates with ACC were considered as ACC deaminase producers ( Supplementary  Fig 23 B, C)